Two new Cortinarius species in subgenus Leprocybe from Southwest China

Two new Cortinarius species in subgenus Leprocybe, Cortinarius hengduanensis and C. yadingensis, are proposed based on a combination of morphological and molecular evidence. Cortinarius hengduanensis has distinct olive tinged basidiomata, a squamulose pileus, and small, subglobose to broadly ellipsoid basidiospores, the ITS sequence differs from that of C. flavifolium by at least 28 substitutions and independent positions. Cortinarius yadingensis has a squamulose pileus and subglobose to broadly ellipsoid coarsely verrucose basidiospores, the ITS sequence has at least 11 substitutions and index position deviations from the other members of the Leprocybe section. Both new species were found in mixed forests of southwest China.


Morphological study
Specimens were collected from the Xizang Autonomous Region, Sichuan and Yunnan Provinces, China.The fresh basidiomata were photographed after collecting from the field and the macro-morphological characters were recorded in detail before drying in an oven at 45 C. A 20% KOH solution was used on fresh pileus and stipe surface, lamella, and context for chemical reaction.Observation of basidiomata was performed under ultraviolet light at a wave length of 360 nm.The specimens were deposited in Fungarium, Institute of Microbiology, Chinese Academy of Sciences (also as Herbarium Mycologicum Academiae Sinicae, HMAS).Descriptions of the microscopical characters are from dried collections.Thin sections were prepared by hand with a razor blade.Sections were mounted in 5% KOH solution.Basidiospores, basidia, tramal hyphae, context, and pileipellis of pileus and stipe were measured using an ocular micrometer.At least 30 basidiospores and 20 basidia of each mature collection were measured.

DNA extraction, amplification and sequencing
Genomic DNA was extracted from dried specimens using standard protocol (Rogers & Bendich, 1994).The DNA extracts were used as templates for PCR.Amplification reactions were performed to obtain sequences of nuclear ribosomal internal transcribed spacer region (ITS) using primer pairs ITS5/ITS4 (White et al., 1990).The amplification was carried out under the following conditions: initial denaturation for 3 min at 95 C,

Phylogenetic analysis
The newly generated ITS sequences were submitted to GenBank.The ITS sequences for the phylogenetic analyses were selected based on results of BLASTn (>94% identity) in GenBank.Two species, C. veronicae (KC017355) and C. veronicoides (GQ890324), were chosen as outgroup.Seventy-three sequences (Table 1) were aligned and edited with BioEdit 7.2.2 (Hall, 1999).Bayesian inference (BI) and maximum likelihood (ML) methods were implemented to analyses in this study.MrModeltest 2.3 was used to calculate the best model (HKY+I+G) for BI analysis (Nylander, 2004).The BI analysis was performed with MrBayes 3.2.6 (Ronquist & Huelsenbeck, 2003), and the ML analysis was conducted in MEGA X (Kumar et al., 2018).The matrix contained 75 ITS sequences with 681 nucleotide sites is available from GenBank (see Data Availability section).Trees were viewed in FigTree 1.4.4 and processed in Adobe Photoshop 2019.

Nomenclature
The electronic version of this article in Portable Document Format (PDF) will represent a published work according to the International Code of Nomenclature for algae, fungi, and plants, and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone.In addition, new names contained in this work have been submitted to Index Fungorum from where they will be made available to the Global Names Index (https://gni.globalnames.org/).The unique Index Fungorum number can be resolved and the associated information viewed through any standard web browser by appending the Index Fungorum number contained in this publication to the prefix "https://www.indexfungorum.org/Names/Names.asp".
The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed, Central SCIE, and CLOCKSS.

Molecular phylogeny
The ITS matrix for phylogenetic analyses included 73 sequences, representing 15 species.The resulting alignments were deposited at TreeBASE (http://www.treebase.org;submission ID 30908; accessed on 1 November 2023).The BI and ML trees showed similar topologies, and the ML tree was selected as the representative phylogeny (Fig. 1).Etymology:-"hengduan", Chinese, referring Hengduan Mountains of southwest China, the locality of the type collection.

DISCUSSION
According to Bidaud et al. (2021), Ammirati et al. (2021) and Liimatainen et al. (2022), subgen.Leprocybe was re-defined as Cortinarius species with small-to medium-sized (occasionally large-sized) basidiomata, obvious UV fluorescent reaction, tomentose to squamulose pileus and subglobose basidiospores, characteristics found in both C. hengduanensis and C. yadingensis.Cortinarius hengduanensis has olive coloration, which was also found in most of the species following: sect.Fuscotomentosi, sect.Leprocybe, sect.Melanoti, sect.Squamiveneti and sect.Veneti (Bidaud et al., 2021;Ammirati et al., 2021).Cortinarius yadingensis does not display any olive tint, its pileus and stipe surfaces are covered by densely dark brown fibrillose squamules, which are found in a few Leprocybe species lacking olive coloration, such as C. pescolanensis (Picillo & Marchionni, 2016).However, phylogenetic analyses of the present research does not support close relationship between C. yadingensis and C. pescolanensis.Ecologically, the two new species are reported in subalpine and alpine areas of southwest China mainly associated with Picea sp. and Quercus sp., but the related members of Leprocybe are mostly distributed in North America or Europe.
Compared with the known diversity of Leprocybe in North America and Europe (Bidaud et al., 2021;Ammirati et al., 2021), only three species were previously reportedfrom China.Cortinarius cotoneus was reported by Keissler & Hohwag (1937) in Yunnan according to a collection of 1914.Horak (1987) reexamined the specimen and confirmed its morphological identification.Shao & Xiang (1997) reported C. cotoneus in Heibei, Shaanxi, Sichuan, Guangdong and C. venetus in Heilongjiang,but these are lacking specimen citations.Sequences from materials of China and East Asia belonging to this lineage with doubtful annotations in GenBank were also included in the phylogenetic analysis (GenBank IDs: HM105543, KX444284, KX513578, KX513580, KX513584-KX513586, LC373240).These sequences nest near C. cotoneus and C. venetus in the phylogenies and may thus represent some unknown species in the section.Unfortunately, the sequenced Chinese materials (e.g., HMAS260331, HMAS254210, HMAS274611, HMAS274352 and HMAS268596) are dried specimens without any in situ photos, which cannot provide sufficient morphological characteristics for the introduction of new species.Xie (2022) reported Cortinarius nigrosquamosus and identified it as a member of Leprocybe foryellowish to olivaceous basidiomata with black squamules.Although the ITS sequence of the studied specimens is the same, there are some morphological variations among the three specimens.Due to these unanswered questions on all those three Leprocybe species historically reported in China, more samplings are still needed to accumulate sufficient data to clarify the diversity of Leprocybe in China.

CONCLUSION
Based on morphological and molecular evidence, Cortinarius hengduanensis and C. yadingensis, were proposed in Cortinarius subgenus Leprocybe.Both species were reported in mixed forest of Southwest China.Cortinarius hengduanensis has distinct olive tinged basidiomata, squamulose pileus, and small, subglobose to broadly ellipsoid basidiospores.Cortinarius yadingensis has a squamulose pileus and stipe, subglobose to broadly ellipsoid, coarsely verrucose basidiospores.Further research on the Leprocybe diversity in China is needed for the likely occurrence of additional new species in the country.
It differs from C. flavifolius for having greenish tint on pileus surface and differs from C. venetus by its obviously annulate stipe.ITS sequences of the new species (GenBank ID: KX513581-KX513583, OR538887 and OR538888) deviate from that of C. flavifolius by at least 28 substitutions and indel positions.In mixed forest with Pinus yunnanensis, P. densata and Quercus aquifolioides.

Table 1
ITS sequences of subgen.Leprocybe used in phylogenetic analysis.